Angiotensin converting enzyme (ACE) is a zinc dipeptidylcarboxypeptidase that is involved in the post-translational processing of bioactive peptides. Endothelial ACE is a key participant in blood pressure regulation and inhibitors of the enzyme are widely employed for treating hypertension. ACE, or ACE-like enzymes, have also been found in a variety of different tissues, notably kidney, heart and brain, where their function is unknown. Moreover, a unique isoform of ACE is present in testis where it is thought to play a role in sperm maturation but how it does this is also unknown. Recently endothelial ACE has been shown to be composed of two homologous domains, each containing a potential zinc binding region/catalytic site, whereas the testicular enzyme has single domain identical to the C-terminal half of the endothelial ACE. We intend to determine whether or not a) each of the two domains of endothelial ACE is catalytically active, b) they are equally active with the same chloride dependence and substrate specificity, c) they interact with each other, d) there are tissue-dependent differences in substrate specificity, e) there are species-dependent differences in activity, and f) there is a chemical or structural basis for such differences. We will also determine the functional role of active site residues in testicular ACE and their relationship to substrate specificity. Toward these ends we will develop methods for expressing endothelial ACE in CHO cells with the intent to examine the role of specific histidine residues in each of the two putative metal-binding regions by site-directed mutagenesis. We will also use this system to express separately the N-terminal and C- terminal halves of the enzyme, and to introduce specific residues and/or segments uniquely present in the C-terminal half of the molecule (which are thought to be involved in catalysis) into the corresponding positions in the N-terminal half. We will use chemical and enzymatic cleavage to separate the N- and C-terminal domains of the somatic enzymes; express, crystallize and carry out mutagenesis of the structurally less complex testicular ACE with principal interest in a putative third metal ligand, a single cysteine residue and the active site glutamic acid; and extend kinetic studies of the unusual endopeptidase activity of endothelial ACE toward luteinizing hormone-releasing hormone (LH-RH) which point to a role for the second domain in the cleavage of this peptide. ACE activity in brain and cardiac tissue reportedly differs from that of somatic or testicular ACE, possibly due to post-translational processing. We will isolate the enzymes from these sources, characterize them kinetically and, if there are differences, determine their structural basis.